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1.
Mol Biol Rep ; 50(4): 3539-3546, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36787053

RESUMEN

BACKGROUND: The co-administration of several therapeutic oligonucleotides targeting the same transcript is a beneficial approach. It broadens the target sites for diseases associated with various mutations or splice variants. However, little is known how a combination of antisense oligonucleotides (ASOs), which is one of the major modalities of therapeutic oligonucleotides, affects the potency. In this study, we aimed to elucidate the combination-effects of ASOs and the relationship between the target sites and potency of different combinations. METHOD AND RESULTS: We designed 113 ASOs targeting human superoxide dismutase 1 pre-mRNA and found 13 ASOs that had comparable silencing activity in vitro. An analysis of combination-effects on the silencing potency of 37 pairs of two ASOs on HeLa cells revealed that 29 pairs had comparable potency to that of two ASOs; on the other hand, eight pairs had reduced potency, indicating a negative impact on the activity. A reduced potency was seen in pairs targeting the same intron, exon-intron combination, or two different introns. The sequence distance of target sites was not the major determinant factor of combination-effects. In addition, a combination of three ASOs preserving the potency could be designed by avoiding two-ASO pairs, which had a reduced potency. CONCLUSIONS: This study revealed that more than half of the combinations retain their potency by paring two ASOs; in contrast, some pairs had a reduced potency. This could not be predicted only by the distance between the target sites.


Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos , Humanos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Células HeLa , Exones/genética , Precursores del ARN
2.
Biochem Biophys Res Commun ; 523(3): 795-801, 2020 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-31954521

RESUMEN

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.


Asunto(s)
ARN Helicasas DEAD-box/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , ARN Helicasas DEAD-box/metabolismo , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Factor 4A Eucariótico de Iniciación/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Bibliotecas de Moléculas Pequeñas/química
3.
Biochem Biophys Res Commun ; 483(1): 271-276, 2017 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-28025139

RESUMEN

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.


Asunto(s)
Empalme Alternativo , ARN Helicasas DEAD-box/química , Regulación Neoplásica de la Expresión Génica , Receptores Androgénicos/genética , Animales , Línea Celular Tumoral , Silenciador del Gen , Variación Genética , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/metabolismo , ARN/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Transcriptoma
4.
Bioorg Med Chem ; 25(7): 2200-2209, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28283335

RESUMEN

Eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase, is a core component of exon junction complex (EJC). EJC has a variety of roles in RNA metabolism such as translation, surveillance, and localization of spliced RNA. It is worthwhile to identify selective eIF4A3 inhibitors with a view to investigating the functions of eIF4A3 and EJC further to clarify the roles of the ATPase and helicase activities in cells. Our chemical optimization of hit compound 2 culminated in the discovery of ATP-competitive eIF4A3 inhibitor 18 with submicromolar ATPase inhibitory activity and excellent selectivity over other helicases. Hence, compound 18 could be a valuable chemical probe to elucidate the detailed functions of eIF4A3 and EJC.


Asunto(s)
Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Historia Medieval , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray
5.
Commun Biol ; 2: 165, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31069274

RESUMEN

The RNA helicase EIF4A3 regulates the exon junction complex and nonsense-mediated mRNA decay functions in RNA transcript processing. However, a transcriptome-wide network definition of these functions has been lacking, in part due to the lack of suitable pharmacological inhibitors. Here we employ short-duration graded EIF4A3 inhibition using small molecule allosteric inhibitors to define the transcriptome-wide dependencies of EIF4A3. We thus define conserved cellular functions, such as cell cycle control, that are EIF4A3 dependent. We show that EIF4A3-dependent splicing reactions have a distinct genome-wide pattern of associated RNA-binding protein motifs. We also uncover an unanticipated role of EIF4A3 in the biology of RNA stress granules, which sequester and silence the translation of most mRNAs under stress conditions and are implicated in cell survival and tumour progression. We show that stress granule induction and maintenance is suppressed on the inhibition of EIF4A3, in part through EIF4A3-associated regulation of G3BP1 and TIA1 scaffold protein expression.


Asunto(s)
Ciclo Celular/genética , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/genética , Estrés Fisiológico/genética , Transcriptoma , Regulación Alostérica/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Biología Computacional/métodos , Gránulos Citoplasmáticos/efectos de los fármacos , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Inhibidores Enzimáticos/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Antígeno Intracelular 1 de las Células T/genética , Antígeno Intracelular 1 de las Células T/metabolismo
6.
Cell Chem Biol ; 25(12): 1470-1484.e5, 2018 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-30293940

RESUMEN

Alternative polyadenylation (APA) plays a critical role in regulating gene expression. However, the balance between genome-encoded APA processing and autoregulation by APA modulating RNA binding protein (RBP) factors is not well understood. We discovered two potent small-molecule modulators of APA (T4 and T5) that promote distal-to-proximal (DtoP) APA usage in multiple transcripts. Monotonically responsive APA events, induced by short exposure to T4 or T5, were defined in the transcriptome, allowing clear isolation of the genomic sequence features and RBP motifs associated with DtoP regulation. We found that longer vulnerable introns, enriched with distinctive A-rich motifs, were preferentially affected by DtoP APA, thus defining a core set of genes with genomically encoded DtoP regulation. Through APA response pattern and compound-small interfering RNA epistasis analysis of APA-associated RBP factors, we further demonstrated that DtoP APA usage is partly modulated by altered autoregulation of polyadenylate binding nuclear protein-1 signaling.


Asunto(s)
Poliadenilación/efectos de los fármacos , Poliadenilación/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Transcriptoma/efectos de los fármacos , Línea Celular , Femenino , Homeostasis/efectos de los fármacos , Humanos , Bibliotecas de Moléculas Pequeñas/química , Transcriptoma/genética
7.
Nat Commun ; 8(1): 7, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28232751

RESUMEN

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.


Asunto(s)
Empalme Alternativo/efectos de los fármacos , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Pirimidinas/farmacología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Exones , Perfilación de la Expresión Génica , Genoma Humano , Células HCT116 , Humanos , Imidazoles/síntesis química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/síntesis química , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Relación Estructura-Actividad , Transcripción Genética
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